Stabilized erythrocyees and methods therefore

ABSTRACT

A method for obtaining an improved aldehyde-treated erythrocyte preparation having an increased hemagglutination titer. The erythrocytes are stabilized by sequential exposure to dilute solutions of pyruvic aldehyde and formaldehyde.

United States Patent Hirata 1 1 Jan. 30, 1973 [54] STABILIZEDERYTHROCYEES AND OTHER PUBLICATIONS METHODS THEREFORE Ling, Brit. J.Haemat. Vol. 7, 1961 pp 2.99302 Inventor? Arihul' Atsunobu "Irma, Kabat,Exptl. lmmunochem. CC Thomas, Springfield Regan, I11. 111 2nd Ed. 1961pp 542-550 [73] Assignee: Abbott Laboratories, North Chem VOL 55 P27495; VOL 65 Chicago, lll. pp 20497, 20672 l Filed: Feb, 1971ngraham,PSEBM,Vol 99,N0v l958,pp 452 56 [21] Appl. No.: 117,790

Primary Exammer-Albert T. Meyers Related U.S. Application Data AssistantExaminerA. P. Fagelman [63] Continuation-impart of Ser. No. 760,052,Sept. 16, Nblack I968, abandoned, which is a continualion-in-part ofSer. No. 682,550, Nov. 13, 1967, abandoned.

[57] ABSTRACT [52] U.S. Cl. ..424/3, 424/11, 424/12,

424/13 252/408 A method for obtammg an improved aldehyde-treated 511lnt.Cl......A0ln 1/00, 00111 31/00, 00111 33/16 erythrocyte Preparationhaving an increasfid hemag- 53 Field f Search 424 3 12 11 13;252 40gglutination titer. The erythrocytes are stabilized by sequentialexposure to dilute solutions of pyruvic al- [56] References Cited dehydeand formaldehyde.

UNITED STATES PATENTS 5 Claims, N0 Drawings 3,057,775 l0/l962 Rendon..424/3 UR STABILIZED ERYTIIROCYEES AND METHODS THEREFORECROSS-REFERENCE TO RELATED APPLICATIONS This application is acontinuation-in-part of copending U.S. Ser. No. 760,052, filed Sept. l6,1968, now abandoned, which is a continuation-in-part of Ser. No.682,550, filed Nov. 13, 1967, now abandoned.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a testpreparation in which erythrocytes for direct hemagglutination testingare provided which have increased activity and greatly improved storagestability. It further relates to a test preparation in which coatederythrocytes for indirect or passive hemagglutination testing areprovided with enhanced activity and very superior storage stability. Itfurther provides a method for treating the erythrocytes to impartthereto these beneficial properties.

Native erythrocytes and those treated by prior art procedures useful fordirect hemagglutination testing such as blood typing suffer from thedisadvantage that they deteriorate rapidly on storage so that for manypurposes they are useless after about 21 days. Moreover, native cellsare not susceptible to freezestorage except in special media, nor arethey satisfactory after being lyophilized and reconstituted. Nativeerythrocytes also are sensitive to the ionic composition, pH and osmoticpressure of the suspending media, so that there exist undesirablerestrictions on the scope of the tests which may be performed with them.

Erythrocytes which are coated with known antigens are a recognizedpreparation for use as a testing means for antibodies specific to suchantigens. When an antiserum or a serum containing the suspected antibodyis brought into contact with the coated erythrocytes, agglutination orclumping results which is an index that the suspected antibody isactually present in the serum. It is known in the art that suchagglutination provides semi-quantitative information as to the presenceand occurrence of specific antibodies and also useful information as toconditions which give rise to the presence of such antibodies.

Many problems have been encountered in preparation, storage and use ofsuch coated erythrocytes. For example, to overcome coating problemsduring praparation, the art has employed coupling agents to applyantigens to the erythrocytes. The problems may also relate generally tothe stability of the erythrocytes under storage conditions to theirsensitivity in selected passive hemagglutination tests and to thereproducability of such hemagglutination testing. Moreover, theindividual erythrocyte cells may clump or exhibit nonspecifichemagglutination, an activity which interferes with hemagglutinationtesting for a specific factor. Coated erythrocytes are ordinarily storedin a frozen condition, but when frozen preparations are thawed, clumpingmay occur or the cells may be destroyed by hemolysis.

it is desirable that erythrocytes be treated so that a variety ofantigens may be coated thereon, and that such coated erythrocytes may beusefully employed as a sensitive reagent for passive hamagglutination,the testing of which is reliable and reproductable. It is also desirableto obtain antigen adsorption without requiring the presence of couplingagents or other involved steps.

It is accordingly one object of this invention to provide an erythrocytepreparation which has been treated so that it can be stored for extendedperiods without resulting in any hemolysis or clumping of the cells. Itis a further object to provide an erythrocyte preparation useful fordirect hemagglutination testing.

A further object of this invention is the provision of a method oftreating erythrocytes to impart storage stability thereto-whilemaintaining the combining sites in active condition.

Another important object of this invention is an erythrocyte preparationof the foregoing type which can be effectively coated with variousantigens, and which coated erythrocytes comprise a preparation which canbe effectively packaged and stored for extended periods until subjectedto particular passive hemagglutination testing.

Still another important object of this invention is to provide a methodfor preparing stable and improved erythrocyte preparations of the typedescribed; and for potentiating such preparations. The term potentiationrefers to the conditioning of cells so as to increase thehemagglutination titer of the preparation.

In accordance with the foregoing objects as well as still other objectswhich will occur to practitioners, the invention is now described indetail in the following disclosure.

The following flow diagram may be referred to for a summary of thefeatures encountered in the practice of the invention.

Contact native erythrocytes with pyruvic aldehyde Contact pyruvicaldehyde-treated erythrocytes with formaldehyde Potentiate stablealdehyde-treated erythrocytes Direct Hem-agglutination Tests Coat stablealdehydetreated erythrocyte preparation with an antigen Coat stablealdehydetreated erythrocyte preparation with an antibody Potentiatestable and coated erythrocytes Passive specific hemagglutination testsPackage and store stable and coated preparations by freezing or bylyopliilization having substantially neutral pH to prevent hemolysis ofthe erythrocytes and to remove undesirable materials which may bepresent on the erythrocytes from the serum.

A suspension of the washed erythrocytes is prepared in the neutralbuffering solution in a minor concentration of preferably less thanabout 10% v/v. The erythrocytes are treated with a minor amount,preferably from 1.5 to 5.0 percent by volume of pyruvic aldehyde,relative to the volume of erythrocytes, for a time sufficient to impartdesirability to the erythrocytes, preferably from 12 to 24 hours withmixing. Following the pyruvic aldehyde treatment step, the erythrocytesare washed several times to remove excess pyruvic aldehyde and any othermaterials which may have originated from the serum. The pyruvicaldehyde-treated erythrocytes are then exposed to a minor amount,preferably from 1.5 to 5.0 percent by volume, of formaldehyde relativeto the volume of erythrocytes, for from about 12 to about 24 hours andthe excess formaldehyde is removed by a plurality of washings with abuffered solution.

The foregoing pyruvic aldehyde and formaldehyde treatments may becollectively referred to as a double aldehyde treatment" to attain astabilized erythrocyte preparation." It is an important and preferredfeature of this invention that the pyruvic aldehyde treatment precedethe formaldehyde treatment because it has been found that with manyerythrocytes an initial formaldehyde treatment will tend to damage suchcells; the preferred sequence is, therefore, recommended as the usualpractice. It is understood, however, that with some erythrocytes, aninitial formaldehyde treatment may not be particularly contraindicatedand, therefore, in some practices an initial formaldehyde treatmentmight still lead to advantageous results if such a sequence ispreferred.

Stabilized erythrocyte preparations may be stored for substantialperiods without undesirable hemolysis, clumping, or other occurrence ofobjectionable features.

Human erythrocytes can be stabilized by pyruvic aldehyde-formaldehydetreatment without apparent alteration in their capacity to react withblood typing antisera. Stabilized human type A, B or AB cells wereagglutinated specifically by anti-A or anti-B serum. The sensitivity ofthe agglutination reaction using the stabilized cells was equal to orseveral fold higher than that using the native cells. Stabilized typecells did not react with anti-A or anti-B serum. The above reactivitieswere retained for several months when the cells were stored at 4C or infreeze-storage. These stabilized cell preparations are useful asreference cells for blood typing.

The foregoing stable erythrocyte preparation may be converted to acoated stable erythrocyte preparation,

by contacting a sample of the aldehyde-treated erythrocytes with anantigen, followed by one or more washing steps to remove access antigen.In general, the stabilized erythrocytes are coated with antigens in asolution buffered to a pH below 7 and desirably buffered to a pH fromabout 3.6 to about 6. For polysaccharide antigens such as E. coliendotoxin, coating can be performed at pH 7. Superior sensitivityresults when the treated erythrocytes are conditioned by agitating themin the buffered solution for about 1 hour prior to coating with antigen.The coating step is usually completed in about 1 hour, but some coatingsmay require as long as 24 hours. A coating temperature of 24C ispreferred; temperatures of 50C and higher produce erythrocytepreparations of reduced sensitivity.

These foregoing conditions may vary somewhat for different antigens, butthe practitioner may readily determine the preferred coating conditionsby reference to agglutination occurrence with serial dilutions of aselected antiserum. For example, a rabbit may be injected with variousamounts of antigen which is identical to the antigen adsorbed on theerythrocyte preparation. A serum sample would be collected in which theantibodies would be present, and serial dilutions of this serum could beplaced in V-bottom plastic trays to which an equal volume of the stablealdehydetreated erythrocyte preparation would be added. A reactionmixture containing cells and diluent serves as a control in which thecells form a button at the bottom, a negative reaction. The appearanceof a matted surface would be a positive reaction of agglutination whichis indicative of satisfactory coating. Non-specific agglutination mustbe carefully distinguished.

When reference is made herein to antigen" coating, such term should beconstrued as denoting a material which may be an antibody or an antigenunless particular reference is made to specific antigens or antibodies.

Representative antigens which may be successfully used to coat thestabilized erythrocyte preparations include protein antigens such asbovine serum albumin or BSA, insolubilized BSA, human serum albumin,rabbit -y-globulin, human y-globulin, streptococcal M protein, ragweedantigen E, polysaccharide antigens such as E. coli endotoxin (ET),acetylated ET, succinylated ET, de-esterified ET, de-esterified andbromacetylated ET, synthetic polyglucose, miscellaneous antigens such asRNA-bacteriophage (Ribonucleic acid), QB, Entamoeba histolytica antigenand still others. It is a particular advantage of this invention thatthe stabilized erythrocyte preparation is suitable for coating with sucha variety of substances with diverse chemical compositions.

ln one embodiment of the potentiating step of this invention, thehemagglutination titer of either the stabilized or the coatederythrocyte preparation may be increased by reducing its temperature toabout lC and then maintaining it at a temperature several degrees belowfreezing, preferably at a uniformly maintained temperature and at about20C, for a period of about 16 weeks. Alternatively, an equally effectivepotentiation results within about 6 weeks by periodically raising thetemperature of the stabilized or coated erythrocytes to 24C for severalhours and without agitation preferably at intervals of about 6 or 7days. Cycles having a length of several hours to a few days wereineffective.

In another embodiment of the potentiating step, the erythrocytes weretreated with a dilute solution of oxidizing agent, sodium periodatebeing a suitable reagent.

In still another embodiment of this invention the stabilized erythrocytepreparation may be potentiated by coating the cells thereof withfibrinogen prior to coating with the antigen. Bovine fibrinogen is asuitable potentiating agent.

The potentiated stabilized or coated erythrocyte preparation may bestored in a lyophilized state or at a temperature of about l90C untilneeded for hemagglutination tests. The manner of performing these testsis well understood by those skilled in-the art.

in general, an animal serum sample with the suspected antibody iscombined with the coated erythrocyte in a liquid vehicle to determine ifany agglutination occurs. Likewise, the stabilized erythrocytepreparations may be coated with a specific antibody so that insubsequent hemagglutination testing, the erythrocytes will agglutinatein the presence of a free specific antigen. Other varieties andmodifications in the hemagglutination testing process will occur topractitioners and all such tests may practiced to advantage with theimproved stable and coated erythrocyte preparations.

The passive hemagglutination test may be used to detect the presence of'y-globulin on the cells as in Coombs test, detection of bacterialinfection as in the presence of endotoxin antigen, detection of viralinfection, the typing for histocompatibility, the detection ofautoimmune diseases and other phenomena of this type. The coated andstable erythrocyte preparations may also be used as a testing reagent inchemical analyses to detect presence of antigen in a quantitative orsemi-quantitative fashion.

The following examples are presented to teach various embodiments forpracticing the invention, but while such embodiments include those bestcontemplated at present for practicing the invention, it should beunderstood that they do not represent exclusive embodiments. Neither dothey necessarily include what may prove to be preferred forms in futurepractice.

EXAMPLE 1 Stable Aldehyde Treated Erythrocyte Preparation A 20 ml.sample of whole blood is removed from a 2-3 kg. rabbit by cardiacpuncture, and said sample is collected in a syringe containing an equalvolume of sterile Alsevers solution. This solution is generally isotonicand contains a sodium citrate anticoaggulant, sodium chloride, dextroseand distilled water. The withdrawn sample is allowed to stand overnightat about 4C, and it is then centrifuged at 1500 rpm. for minutes toobtain about 10 ml. of packed rabbit erythrocytes. The packederythrocytes are then combined with 10 volumes of a 0.11M phosphatebuffer solution, pH 7.2. This buffered solution is prepared frommonopotassium phosphate and disodium phosphate with vortex mixing. Themixture of erythrocyte and buffer solution is centrifuged and thensubjected to five separate washings with 10 volumes of the foregoingbuffered solution to remove serum protein and any other serum materials.The washed erythrocytes are then resuspended in the foregoing bufferedsolution to attain an 8% v/v rabbit erythrocyte suspen- SlOll.

To a 500 ml. Erlenmeyer flask is added 125 ml. of the phosphate bufferedsolution containing 3 percent pyruvic aldehyde v/v and 125 ml. of theforegoing 8 percent erythrocyte suspension, and the mixture is stirredabout 18 hours at room temperature. The mixture is then filtered througha gauze pad to remove any large debris andthe erythrocytes are thenwashed on five separate occassions with 10 times their volume of thephosphate buffered solution. The pyruvic aldehyde treated erythrocytepreparation is then resuspended in the foregoing buffered solution in a10 percent concentration v/v.

A 125 ml. volume of the foregoing pyruvic aldehyde treated suspension isdiluted to 8 percent and mixed with 125 ml. of the phosphate bufferedsolution containing 3 percent formaldehyde v/v. The mixture is agitatedovernight with a magnetic stirrer at room temperature, and the mixtureis then filtered through a gauze pad to remove any possible debris. Thepyruvic aldehyde and formaldehyde treated erythrocytes are then washedon five different occassions with 10 times their volume of the foregoingbuffered solutionThe aldehyde treated erythrocyte preparation is thenresuspended as a 10 percent concentration in the foregoing bufferedsolution and transferred to glass containers and stoppered for storage.The foregoing aldehyde treated erythrocyte preparation is stable for atleast 2 months at reduced temperatures of 4C, or for indefinite periodsfrozen in liquid nitrogen.

The stabilized erythrocyte preparation may be potentiated, but in thepreferred process the potentiation step is reserved until after theerythrocytes have been coated.

The stabilized erythrocyte preparation made according to the process ofthis invention possesses most, if

' not all of the combining sites present on the native erythrocyte,combining sites referring specifically to sites which may react withantibody or virus.

EXAMPLE 2 Potentiated Stabilized Erythrocyte Preparation This examplewill illustrate the increase in hemagglutination titer achieved byattaching fibrinogen to the cells of the stabilized erythrocytepreparation. Bovine fibrinogen at a concentration of l'microgram permilliliter in 0.1 M acetate buffered solution, pH 4.0, was heated at 60Cfor one hour and cooled to room temperature. A l0 percent suspension ofthe double aldehyde treated erythrocytes obtained by the process ofExample 1 were added to the fibrinogen solution and stirred for one hourat room temperature. The potentiated stabilized erythrocytes were washedwith a phosphate buffer solution, pH 7.2, and the erythrocyteconcentration adjusted to 10% v/v. These cellswere later used withsuperior results for blood type determinations.

EXAMPLE 3 Potentiated, Stabilized, Coated, Erythrocyte Preparation Onemilliliter of the 10 percent suspension of potentiated stabilizederythrocyte preparation obtained by the process of Example 2 was washedwith a 0.1 M'

acetate buffer solution, pH 4.0, and resuspended in 9 milliliters of theacetate buffer solution at 24C. One milliliter of the acetate buffersolution containingl milligram of BSA is added. The mixture was stirredat room temperature for 2'hours and then washed 5 times with an excessof phosphate buffered solution, pH 7.2. These cells were later used forpassive hemagglutination reactions.

EXAMPLE4 Antigen Coated Stabilized Erythrocyte Preparation lnto a 12 ml.conical centrifuge tube at 24C is added 1 ml. of the 10 percentsuspension of aldehyde treated erythrocyte preparation obtained by theprocess steps of Example 1. This sample is then washed with a 0.1 molaracetate buffer solution, pH 4.0. The stabilized erythrocytes are nothemolized or clumped by this lowered pH level. The washed erythrocytepreparation cells are resuspended in 9 ml. of the acetate bufferedsolution at 24C, pH 4.0 and agitated at this pH and temperature for 1hour; then 1 ml. of the acetate buffer solution containing 1 mg. of BSAis added. The mixture of antigen and stable aldehyde treatederythrocytes are rotated at room temperature for 2 hours and thensubjected to 5 separate washings with excess volumes of a phosphatebuffered solution, pH 7.2. The BSA coated stable erythrocytes areresuspended in the foregoing phosphate buffer at concentrations of 0.5%v/v, and I ml. aliquots of this suspension are packaged in 20 ml. glassbottles and stoppered. The packaged predetermined amount of the coatedstable aldehyde treated erythrocyte preparation is then quick frozen ina liquid nitrogen freezer and stored at 20C.

EXAMPLE Potentiation of Coated Stable Erythrocyte Preparation Treatederythrocytes, coated as in Example 4, but at a pH of 3.6, having beenfrozen at l96C and stored at 20C for 6 days, are thawed to roomtemperature of 24C without agitation, maintained at 24C for 2 hours, andafter refreezing at l96C, are returned to 20C. The process is repeatedonce a week for 6 weeks.

The potentiated coated erythrocytes are then frozen at 196C and storedat that temperature until needed for the hemagglutination test.Alternatively, the potentiated coated cell suspension can be lyophilizedand reconstituted by adding water before use.

This process increases the titer 30-fold while only doubling thenon-specific hemagglutination titer, a practically insignificantincrease. Further repeated freeze-thaw cycles results in a furtherincrease in hemagglutination titer but also produces a concurrentsignificant increase in the nonspecific hemagglutination titer, so thatlittle advantage is realized from the extended processing.

The optimum number of freezethaw cycles is related to a specific coatedcell preparation but the optimum will be recognized by those skilled inthe art as occurring at that cycle beyond which the increase inhema'gglutination titer is accompanied by an appreciable increase in thenon-specific hemagglutination titer. A coated cell preparation which wasoptimal for the standard serum was found to be optimal also for otherantisera. Thus, successive standard sera can be standardized against theoriginal selected anti-serum.

EXAMPLE 6 Sensitivity of Erythrocyte Preparation Coated with BovineSerum Albumin Following the procedures of Examples l and 4, anerythrocyte preparation is obtained in which rabbit erythrocytes arecoated with Bovine Serum Albumin (BSA). A related procedure is followedto coat another erythrocyte preparation with BSA, but the erythrocytesofthis sample are stabilized only with pyruvic aldehyde and not with thealdehyde treatment sequence according to the disclosure of thisinvention. The hem-agglutination titers of both preparations arecompared in the following Table l to illustrate the greater sensitivityof the BSA coated erythrocyte preparation which has been stabilized withthe double aldehyde treatment. Also illustrated are the results of thepotentiation step.

EXAMPLE 7a Potentiated Stabilized Erythrocyte Preparation This examplewill illustrate still another embodiment of the potentiating step ofthis invention. Human erythrocytes were processed according to theprocedure of Example 1 to obtain stabilized human erythrocytes suspendedin a phosphate buffered solution, pH 7.2. The stabilized humanerythrocytes were then treated at 24C for 15 minutes with 0.00035Msodium periodate solution and then washed free of this reagent withphosphate buffered solution pH 7.2. When subjected to directhemagglutination reaction against rat antiserum, the stabilized cellsgave a reaction at dilution of l to whereas the stabilized cellspotentiated with the oxidation reaction exhibited hemagglutination at adilution of l to 2,000.

EXAMPLE 7b Potentiated, Stabilized Coated Erythrocyte Preparation Anantigen coated stabilized erythrocyte preparation obtained according tothe process of Example 4 was treated as above with 0.00035 M sodiumperiodate for 15 minutes at 24C. The potentiated stabilized erythrocyteswere washed with a phosphate buffered solution pH 7.2 and found to havea hemagglutination titer of 1 to 2,400,000.

TABLE I Comparison of Hemagglutination Reaction of ErythrocytePreparations Condition Duration (Hours) Hemagglutina- Coating tion TiterCoated Cells pH EXAMPLE 8 Representative Variations in Conditions ForCoating Aldehyde-Treated Erythrocyte Preparations with Various AntigensA number of representative antigens are selected for coating anerythrocyte preparation prepared according to the procedure set out inExample 1. The coating procedure of Example 4 is generally followed.

The following data illustrates recorded observations which are readilydetermined by skilled practitioners by noting the occurrence of positiveagglutination with serial dilutions of rabbit serum as previouslydescribed.

Plus one hour agitation at pH 4 prior to antigen addition. Plus one houragitation at pH 3.6 prior to antigen addition.

The invention may now be practiced in the many various ways which occurto those skilled in this art, and all such modifications in practicewill comprise a part of the concept behind the disclosed embodiments.

The invention is now defined by the terms of the following claims whichare given further meaning by the language of the preceding description.

I claim:

1. A method for obtaining an improved aldehydetreated erythrocytepreparation which is useful in passive hemagglutination procedures, saidmethod comprising the steps of: preparing a suspension of washederythrocytes in a neutral buffered solution; treating said erythrocytesuspension with from 1.5 to 5.0 percent by volume of pyruvic aldehydeper volume of erythrocytes for from 12 to 24 hours at room temperature;washing the pyruvic aldehyde-treated erythrocyte suspension with saidbuffered solution to remove the excess pyruvic aldehyde; treating thewashed pyruvic aldehyde-treated erythrocyte suspension with from 1.5 to5.0 percent by volume of formaldehyde per volume of erythrocytes forfrom 12 to 24 hours; and washing the resulting stabilized erythrocytepreparation with said buffered solution to remove the excessformaldehyde.

2. The method of claim 1 wherein said neutral buffered solution is a pH7.0-7.2 phosphate solution.

3. The method of claim 2 additionally comprising the step of treatingsaid stabilized erythrocyte preparation at about 24C. for about 15minutes with a 0.00035 M sodium periodate solution and then washing saidpreparation with said buffered solution to remove the oxidizing agent.

4. The stable erythrocyte preparation obtained by the method of claim 1.

5. The stable erythrocyte preparation of claim 4 in lyophilized form.

1. A method for obtaining an improved aldehyde-treated erythrocytepreparation which is useful In passive hemagglutination procedures, saidmethod comprising the steps of: preparing a suspension of washederythrocytes in a neutral buffered solution; treating said erythrocytesuspension with from 1.5 to 5.0 percent by volume of pyruvic aldehydeper volume of erythrocytes for from 12 to 24 hours at room temperature;washing the pyruvic aldehyde-treated erythrocyte suspension with saidbuffered solution to remove the excess pyruvic aldehyde; treating thewashed pyruvic aldehyde-treated erythrocyte suspension with from 1.5 to5.0 percent by volume of formaldehyde per volume of erythrocytes forfrom 12 to 24 hours; and washing the resulting stabilized erythrocytepreparation with said buffered solution to remove the excessformaldehyde.
 2. The method of claim 1 wherein said neutral bufferedsolution is a pH 7.0-7.2 phosphate solution.
 3. The method of claim 2additionally comprising the step of treating said stabilized erythrocytepreparation at about 24*C. for about 15 minutes with a 0.00035 M sodiumperiodate solution and then washing said preparation with said bufferedsolution to remove the oxidizing agent.
 4. The stable erythrocytepreparation obtained by the method of claim 1.